The major purpose of this work is to investigate a novel mechanism of gene regulation during development. Specifically, these studies focus on a novel regulatory RNA, embryonic ventral forebrain (Evf). Presently, very little is known about non-coding RNAs (ncRNAs) in development. Evf ncRNAs are the first developmentally regulated ncRNAs to be discovered that affect the transcriptional activity of a specific enhancer. Evf ncRNAs are also the first ncRNAs shown to cooperate and complex with a homeobox-containing transcription factor. The proposed studies would therefore be the first to investigate the in vivo role of developmentally regulated ncRNA-dependent modulation of enhancer activity. If ncRNA-mediated enhancer regulation proves to be a common transcriptional regulatory mechanism, this will have applications reaching far beyond the study of development. Functional regulatory RNAs are thought to be involved in Prader Willi Syndrome, diGeorge Syndrome, Beckwith-Wiedeman syndrome, Spinocerebellar ataxia type 8, and campomelic dysplasia, and thus the proposed experiments will be relevant to understanding how these diseases are caused. We show that Evf ncRNAs regulate the expression of the Dlx 5 and 6 homeobox-containing transcription factors. We also show that the Evf gene overlaps with the Dlx 5/6 genes in a region of synteny between the mouse and human chromosomes 6 and 7, respectively. The importance of the Dlx 5/6 genes and this particular region of mouse chromosome 6 is clear from the recent demonstration that the deletion of Dlx 5/6 in the mouse phenocopies the human genetic disease, split-hand-split foot malformation (SHFM). Therefore, investigation of Dlx 5/6 regulation will be important to understanding craniofacial and limb development as well as a human disease that results from alterations in these processes. [unreadable] [unreadable] [unreadable]